1. Field of the Invention
The present invention relates to a method for obtaining immunoassay measurements for samples having an extended antigen concentration range and for providing a signal at high antigen levels. In particular, the present invention provides a calibration method to provide a linear correlation between two turbidimetric measurements made on samples having a high antigen concentration.
2. Description of the Related Art
Agglutination reactions have long been used in immunoassays to measure the concentration or presence of a wide variety of bacteria, cell-surface antigens, serum proteins or other analytes of clinical interest. Agglutination results from the reaction of bivalent antibodies with multivalent antigens of interest to produce aggregates which can be detected and measured in various ways. Similarly, the same reaction can be utilized for the detection of specific antibodies by the agglutination reaction caused by the addition of the corresponding antigen.
Increased sensitivity to visual or instrumental detection of agglutination or its inhibition can be achieved by the use of particle reagents as carriers, rather than soluble proteins or protein conjugates. Antibody particle reagents are also known. A common method for preparation of such reagents is by adsorption of the antibodies onto the surface of suitable adsorbents. Polystyrene-based latex particles have been used extensively for this purpose.
Immunological techniques for the detection of antigens by reaction with antibodies are known to be adversely affected by the well-known and so-called "hook effect" which introduces nonlinearity in agglutination reactions for sample having at high antigen concentrations. In these reactions, the particle-antibody reagent is provided in sufficient concentration to ensure that sufficient binding sites are available to capture all antigens within the sample. However, patient samples may contain a unusually high concentration of antigens so that all available antibodies within the particle reagent become attached to antigens. In this instance, further increasing antigen concentration results in a increase in immobilized label until the antigen concentration becomes so great that fewer sandwich complexes are formed resulting in an erroneous indication of the antigen concentration actually present in the sample. In these instances, the production of an linear assay calibration curve creates special problems because when the antigen is present in such an "excess" amount, it cannot act as the limiting factor in the reaction, thus producing a highly non-linear calibration curve.
Conventional immunoassay methods have attempted to address the hook-effect problem by supplying greater numbers of both the labeled and immobilized ligand binding partners in order to accommodate greater ligand concentrations. This approach, however, disadvantageously results in greater economic costs and is further constrained if the amount of ligand binding partners which may be immobilized per unit area on a solid phase is limited. Such increased concentrations may also be at the expense of sensitivity since increasing the numbers of binding partners and the area available for attachment of binding partners can result in inefficiencies in forming ligand-binding partner complexes with the few ligands that may be present in a sample having a very low ligand concentration.
Various attempts have been made to produce linear calibration curves in immunoassays which measure antigen concentrations that may not generate a linear reaction. One approach is to adjust the amount of insolubilized particle reagent thereby increasing the antibody concentration so that the antigen becomes the rate-limiting factor in the system; however, there is a limit to the amount of reagent that may be added without adversely affecting assay sensitivity. Another approach is the addition of excess labeled antibody, however, this increases the background level of the standard immunoassay. A third approach manipulates the calibration curve in order to achieve a relatively linear curve using a technique known as logit transformation to produce a semilogarithmic plot of the relationship between absorbance and antigen quantity. In such a plot, the relationship between absorbance and antigen concentration can be approximated by a linear function, in a limited analytical range. While a linear approximation of the transformed curve can be made based on this approach and a conversion factor can be calculated from the data, the necessity of running a full standard curve, rather than a single standard, remains.
U.S. Pat. No. 4,595,661 discloses an improved immunoassay for a antigen in a fluid which comprises contacting the fluid with at least one first entity selected from a group consisting of an antibody (Ab) to the antigen (Ag), a soluble, labeled antibody to the antigen, and an antibody to the antigen bound to a solid carrier. The immunoassay is characterized in that the fluid is contacted with at least one additional entity selected from a group consisting of at least one different type of soluble, labeled antibody to the antigen, at least one different type of antibody to the Ag bound to a solid carrier, and at least one different type of antibody to the Ag. Each of the additional entities has an average affinity constant for the Ag lower than the K of its corresponding first entity for the Ag. In addition, the additional entity is present in an amount sufficient to avoid the hook effect.
U.S. Pat. No. 4,743,542 discloses a method for reducing the hook effect in immunoassays which is especially useful for immunoassay systems for the detection of ligands wherein the order of reactions, volumes of reactants, and number of wash steps are kept constant between ligand assays. The disclosed immunoassay reactants preferably include a haptenated ligand binding partner specific for the ligand to be detected and an insoluble, isotactic surface means for immobilizing said haptenated first binding partner. The reactants further include a reaction component having an enzyme label associated therewith and which becomes associated with said first binding partner in accordance with the presence or absence of the ligand to be detected. The method comprises adjustment of the reactants by addition to the aforedescribed reactants, ligand binding partner without associated hapten or, reaction component without associated enzyme label, or a combination thereof.
U.S. Pat. No. 4,788,138 is a method for improving measurement accuracy in which excess first antibody not bound to the solid support and/or excess unlabelled unbound second antibody are added to the immunoassay system. The first unbound unlabelled antibody acts as an analogue for the first antibody bound to the insoluble support. The first unbound unlabelled antibody may be a different antibody from the first antibody which is insolubilized. The unbound unlabelled second antibody may be a different antibody from the labeled second antibody for which it acts as an analogue. The addition of first antibody not bound to the insoluble support is not limited by the surface area available on the insoluble support. The addition of unlabelled unbound second antibody will not increase the background of the immunoassay. The addition of one or both of these analogues will, however, enable one to achieve a pseudo first-order reaction and, thus, a linear standard curve.
U.S. Pat. No. 5,358,852 discloses a specific binding immunoassay method in which a liquid sample containing C-reactive protein (CRP) in the presence of calcium ions is combined with a first antibody Abl specific for CRP and immobilized on a water-insoluble substrate and a labeled, unbound second antibody Ab2 specific for CRP to obtain a water-insoluble complex of Ab1, CRP, and Ab2. The water-insoluble complex is separated from the liquid sample and unreacted Ab2 followed by measuring either the amount of Ab2 associated with said water-insoluble complex or the amount of unreacted Ab2 as an indication of the amount of CRP in the sample. The presence of calcium ions improves the signal system of a sandwich assay specific to CRP sufficient to avoid the so-called "hook" effect. The CRP may be quantified whether present in the specimen at a very low or a very high concentration without risk of false negative results when CRP is present in high concentrations. Further, the presence of calcium has not been found to interfere with the measurement of CRP when CRP is present in low concentrations in the sample.
Accordingly, from a study of the different approaches taken in the prior art to the problems presented by the necessity for providing an actual linear standard curve in immunoassays designed to detect and/or measure antigen present in sufficient quantity such that prior art adjustments to the immunoassay system fail to yield a linear standard curve, there remains a need for an improved approach to provide liquid samples to an automated clinical analyzer without introducing complex control mechanisms and without unduly adding to the resources required.